The present invention relates to novel pyrrolopyridinium derivatives; an antibody prepared from said derivatives as a hapten; a method for the diagnosis of diseases including diabetes, diabetic complications, amyoidosis, renal failure, dialysis-related complications, aging, and diseases accompanied by aging, such as Alzheimer""s disease, etc. by measuring said derivative or by measuring reactivity in the subject with an antibody prepared therefrom; and a method for evaluating the effectiveness of pharmaceuticals which are effective for treating diabetes, diabetic complications, amyloidosis, renal failure, dialysis-related complications, aging, and diseases accompanied by aging, such as Alzheimer""s disease, etc.
In 1968, glycosylhemoglobin (HbAlc), which is one of the minor components of hemoglobin was identified in vivo and was found to increase in patients diagnosed as having diabetes. With this discovery, the Maillard reaction was shown to occur with proteins in vivo. Despite having been studied mainly in the field of food chemicals, the biological and medical meaning of the Maillard reaction would now receive greatly increased public attention.
The Maillard reaction may be classified into a former stage and a latter stage. In the former stage, a Schiff base is formed by the condensation of an amino group of a protein with an aldehyde group of a reducing sugar and is stabilized as a result of the Amadori rearrangement. In the latter stage, the rearranged Schiff Base is transferred, after a long series of reactions, to an Advanced Glycation Endproduct (AGE). The latter stage end products are characterized by fluorescence, a color change to brown and molecular crosslinking. In recent years, several studies on AGE have investigated the relationship between various diseases and AGE, which is produced particularly in the advanced stages of various diseases.
Several studies have uncovered the participation of AGE in various diseases and in the aging process. See xe2x80x9cAGE (glycation) and Diseasesxe2x80x9d (BIO Clinica, 11(5), p. 314 (1996)), which is a review by Dr. Horiuchi et al. For example, aging is thought to be a process in which various proteins and DNA in tissues are modified or changed until they can no longer be decomposed and removed. The protein and DNA so-modified are accumulated in the body, resulting in a loss of their function. The advanced glycation of tissue proteins is exemplified as one of the causes thereof. In fact, it has been shown that the fluorescence of the hydrolyzed products of skin collagen and lens protein increases with aging and that an excessive advanced glycation of lens proteins participates in the onset of cataracts. In addition, AGE is now being recognized as one of the causes of the aging phenomenon because advanced glycation causes an inhibition of enzymatic activity and a corresponding decrease in the body""s ability to decompose intracellular protein. AGE also causes a substance transport disorder brought on by advancedly glycated cytoskeleton protein and DNA mutation caused by a cross-linkage formed between DNA and protein by AGE.
Studies using an antibody to the above-mentioned AGE have uncovered the participation of AGE in various diseases. Still other reports show that an anti-AGE antibody reacts in a concentration-dependent manner with the water-soluble fraction and the alkali-soluble fraction of the normal human lens crystallin, while a very high correlation is observed between anti-AGE antibody reactivity and the aging process; that advanced glycation in the Descemet membrane and lens vesicle of cattle increases with age, as shown by immune electric microscopy; and that the AGE immunoreaction with the petrosal nerve cell correlates with aging, as shown by an immunohistochemical investigation using an antibody.
Fluorescence, which is characteristic of the presence of AGE, is used as an indicator for investigating the correlation between AGE and various diseases. It is known that in diabetic patients fluorescence is significantly higher than it is in healthy people. Moreover, the accumulation of AGE was reported in renal tissues, such as the renal mesangium region, blood vessel walls and urinary tubules, and in the coronary arteriosclerotic nest of diabetic patients. It was also reported that, as a result of immune staining using an anti-AGE antibody, AGE shows almost the same distribution in the peripheral nerves of both experimental diabetic rats and human diabetics. Accordingly, it has been recognized that there is a correlation between diabetes mellitus and AGE.
It has also been shown that an excessive formation of AGE in an organism is a cause of diabetic complications. For example, an advanced glycation of crystallin, which is a lens protein, is one of the causes of diabetic cataracts. It has also been shown that AGE is deeply related to diabetic nephropathy. The glomerular basement membrane in the kidney, comprised of collagen, is advancedly glycated by hyperglycemia and is apt to be bound to albumin, immunoglobulin, and low-density lipoprotein, etc., whereby the basement membrane thickens, thus disturbing the renal filtration function. In addition, reports suggest that AGE may accumulate in mesangium cells and that it may participate in inflammation, such as the secretion of cytokines mediated by a receptor. Recently, it has been reported that a suppressor for the production of AGE is useful in treating diabetic nephropathy. It has been also reported that the myelin protein of a nerve cell is advancedly glycated, suggesting that AGE participates in diabetic neuropathy as well. From these reports, a clear correlation emerges between increased amounts of AGE in the body and the onset of diabetic complications such as diabetic nephropathy, arteriosclerosis, neurosis, retinopathy, cataracts, etc. AGE and diabetic complications are summarized, for example, in a review entitled xe2x80x9cAGE and Diabetic Complicationsxe2x80x9d (Saishin Igaku, 49, 2, p. 248 (1994)) by Morisaki, et al.
In addition, a relation between AGE and dialysis-related amyloidosis has been found because fluorescence increases in the protein found in the sera of dialyzed patients. A similar accumulation of AGE occurs at the site where a sediment forms in the amyloid of the carpal tunnel of dialyzed patients.
A recent report suggests that a scavenger receptor recognizing the AGE protein is present in monocytes, macrophages, mesangium cells and endothelial cells, and that the AGE recognition mediated by such a receptor results in the release of cytokine, the promotion of blood vessel permeability, abnormal blood flow, etc. A relationship thus emerges between AGE and the symptoms of inflammation, capillary obstruction, arteriosclerosis, etc. Furthermore, it is suspected that AGE participates in the onset and progress of Alzheimer""s disease because AGE is strongly detected in senile plaque, and in amyloid and neurofibril changes which are characteristically noted in the brains of patients suffering from Alzheimer disease.
The Maillard reaction may also generate free radical oxygen. Free radical or active oxygen is receiving much recent public attention as a cause of aging, tissue disorders resulting from diabetes mellitus and the generation of pathological changes resulting from Alzheimer""s disease. It is becoming clear that the Amadori compound produced by the initial-stage reaction of the Maillard reaction is oxidized by active oxygen to produce AGE, while at the same time causing the generation of active oxygen. It has been also reported in neuromatous cell strains (SKxe2x80x94Nxe2x80x94SH) that neurofibril (PHF) tau-protein generates active oxygen by glycation and that it activates the transcription factor NFxe2x80x94xcexaB. It has also been suggested that an extinction enzyme for active oxygen such as Cuxe2x80x94Zn-superoxide dismutase (Cuxe2x80x94Zn-SOD) is advancedly glycated as its activity lowers with aging. Such glycation of Cuxe2x80x94Zn-SOD becomes over time an essential cause for an increase in oxidative stress and suggests again the relation between Alzheimer""s disease and AGE.
Until now, several AGEs such as Nxcex5-carboxymethyllysine (CML), pentosidine, pyralin, Nxcex5-carboxyethyllysine (CEL), glyoxallysine dimer (GOLD) and methylglyoxallysine dimer (MOLD) have been reported.
Antibodies to some of the commercially available AGE compounds have been manufactured and studied. An antibody recognizing such an AGE, 6D12 (Kumamoto Antibody Laboratory) which is an anti-CML monoclonal antibody, has been put on the market. Immunohistological and immunochemical studies, such as the measurement of AGE amounts and immunological staining in human plasma, human serum, human urine, arteriosclerotic tissue, the renal tissue of patients suffering from diabetic nephropathy, the eye tissue of aged people, the cerebral tissue of patients suffering from Alzheimer""s disease, etc., using antibodies of the above-mentioned AGEs have been carried out and studies of their medical significance of existence have been conducted.
However, the fact that a large number of biological Maillard reactions and commercial methods exist which produce AGE suggests that various kinds and types of AGE are produced depending upon each situation and that the distribution of AGE in any tissues will vary widely. It is thus desirable to persons skilled in the art to obtain many AGEs having various different properties for the purpose of finding an optimum AGE suitable for each of the various kinds and types of situations in an organism. For example, the present inventors and their co-workers found a pyridinium derivative called Crossline(trademark) (Japanese Patent Laid-Open Hei-06/73057), naphthylidinium derivatives disclosed in Japanese Patent Laid-Open Hei-08/48686 and corresponding U.S. Pat. No. 5,686,251, and pyrrolonaphthylidium derivatives disclosed in Japanese Patent Laid-Open Hei-10/158265, etc. as novel AGEs. U.S. Pat. No. 5,338,850 to Nakamura, et al and corresponding Japanese Laid Open Hei-06/73057 and corresponding European Patent Publication No. 0695750 disclose pyridinium derivatives which are effective in diagnosis of diabetes, diabetic complications, aging, and diseases accompanied by aging. An antibody prepared from the pyridinium derivatives as a hapten is also disclosed. Evaluation of the effectiveness of pharmaceuticals for the treatment of these ailments is also described. Nevertheless, there remains a demand for a novel AGE produced under a situation that is different from the conventional one or, in other words, for a novel AGE having a structural skeleton different from the conventional AGE.
The present inventors have conducted continued studies on crosslinked substances which are produced by each of the processes of nonenzymatic glycation and the oxidation of proteins for use as an indicator to diagnose diabetic complications, dialysis complications and aging. As a result thereof, they have found novel and unobvious pyrrolopyridinium derivatives which have a completely new structural skeleton from that of known AGEs.
The present invention provides: (a) novel pyrrolopyridinium derivatives and salts thereof; (b) an antibody prepared from said derivatives as a hapten; (c) a method for the diagnosis of diabetes, diabetic complications, dialysis-related complications, amyloidosis, aging, diseases accompanied by aging, etc. using said derivatives or an antigen or antibody thereof; and (d) a method for evaluating effectiveness of pharmaceuticals used to treat diabetes, diabetes-related diseases, dialysis-related complications, aging, diseases accompanied by aging, etc.
The novel pyrrolopyridinium derivatives of the present invention are represented by the following formulae (I), (II) or (III), and steroisomers and enantiomers thereof and pharmaceutically acceptable salts of the derivatives, stereoisomers and enantiomers: 
wherein each of Rxe2x80x2 and Rxe2x80x3 is, independently, an alkyl group which may have an amino, a protected amino and/or a carboxyl substituent.
A method of manufacturing the pyrrolopyridinium derivatives of the present invention may comprise, for example, reacting a compound of the formula Rxe2x80x2NH2 or Rxe2x80x3NH2, wherein Rxe2x80x2 and Rxe2x80x3 are the same groups as mentioned above, with sugars, for example, hexose such as glucose, fructose, galactose, mannose or deoxyglucose, amino sugar such as glucosamine or galactosamine, oligosaccharide such as saccharose, for a sufficient period of time to give the derivatives of the present invention, followed by recovering the derivatives from the reaction mixture. The pyrrolopyridinium derivatives of the present invention can also be manufactured by hydrolyzing the product obtained by reacting a sugar, as defined above, with protein or peptide as amine component.
The novel pyrrolopyridinium derivatives of the present invention are represented by the following formulae (I), (II) or (III), stereoisomers and enantiomers thereof, and pharmaceutically acceptable salts of the derivatives of formulae (I), (II), or (III), stereoisomers and enantiomers thereof: 
wherein Rxe2x80x2 and Rxe2x80x3 is the same or different and each of them is an alkyl group which may have an amino, a protected amino and/or a carboxyl substituent.
Examples of the preferred alkyl for Rxe2x80x2 and Rxe2x80x3 in the above general formula, (I), (II) and (III) are linear or branched alkyl having 1 to 6 carbon atom(s) such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, neopentyl, tert-pentyl, hexyl and dimethylbutyl.
The alkyl groups Rxe2x80x2 and Rxe2x80x3 may be substituted with at least one protected amino group. Protective groups for the amino substituent on Rxe2x80x2 or Rxe2x80x3 include, for example, those which are commonly used in the field of peptide synthesis. These may include, for example, formyl, acetyl, tosyl, benzyloxycarbonyl, p-methoxybenzyloxycarbonyl, p-chlorobenzyloxycarbonyl, p-nitrobenzyloxycarbonyl, p-phenylazobenzyloxycarbonyl, p-methoxyphenylazobenzyloxycarbonyl, tert-butoxycarbonyl (Boc), p-toluenesulfonyl (Tos), tert-amyloxycarbonyl, p-biphenylisopropyloxycarbonyl and diisopropylmethyloxycarbonyl groups.
Among the pyrrolopyridinium derivatives of the present invention represented by general formulae (I), (II) and (III), pyrrolopyridinium derivatives wherein Rxe2x80x2 and Rxe2x80x3 are alkyl having amino and/or carboxyl groups can easily be combined with a carrier protein as haptens useful, in particular, for preparation of the antibody of the present invention. The carrier for combining with a hapten for the preparation of an antibody of the present invention, may include commonly-used carriers such as protein (e.g. serum albumin, keyhole lympet hemocyanin) and polymers (e.g. polylysine).
The pyrrolopyridinium derivatives of the present invention include salts of the derivatives represented by general formulae (I), (II) and (III). These salts may include pharmaceutically acceptable salts with at least one metal, acid or base. Exemplary salts may include salts with acids like hydrochloric acid, sulfuric acid, nitric acid, hydrobromic acid, phosphoric acid, perhydrochloric acid, thiocyanic acid, boric acid, formic acid, acetic acid, haloacetic acid, propionic acid, glycolic acid, citric acid, tartaric acid, succinic acid, gluconic acid, lactic acid, malonic acid, fumaric acid, anthranilic acid, benzoic acid, cinnamic acid, p-toluenesulfonic acid, naphtalenesulfonic acid and sulfanilic acid. Further examples may include addition salts with ammonia or with organic bases such as organic amines; and salts with an alkali metal such as sodium and potassium; salts with an alkali earth metal such as magnesium, calcium and barium; and salts with other metals such as aluminum and zinc.
The salts of the pyrrolopyridinium derivatives of the present invention may be manufactured by conventional methods from the pyrrolopyridinium derivatives of the present invention in a free form and may include mixed salts. Likewise, salts of the pyrrolopyridinium derivatives of the present invention may be readily converted from salt form to a free form by conventional methods.
If the pyrrolopyridinium derivatives of the present invention have stereoisomers such as cis-trans isomers, optical isomers, conformational isomers, or if there are hydrates and/or complexes of the pyrrolopyridinium derivatives of the present invention, the pyrrolopyridinium derivatives of the present invention include any and all of them.
A method of manufacturing the pyrrolopyridinium derivatives of the present invention may comprise, for example, reacting a compound of the formula Rxe2x80x2NH2 or Rxe2x80x3NH2, wherein Rxe2x80x2 and Rxe2x80x3 are the same groups as mentioned above, with sugars, for example, hexose such as glucose, fructose, galactose, mannose or deoxyglucose, amino sugar such as glucosamine or galactosamine, oligosaccharide such as saccharose, for a sufficient period of time to give the derivatives of the present invention, followed by recovering the derivatives from the reaction mixture. The pyrrolopyridinium derivatives of the present invention can also be manufactured by hydrolyzing the product obtained by reacting a sugar, as defined above, with protein or peptide as amine component.
There is no particular restriction on the reaction conditions such as reaction temperature, reaction time, pH, etc. and they may be readily established. For example, an easy procedure comprises allowing the reaction mixture to stand at room temperature and at atmospheric pressure, however, the reaction can be accelerated by heating and the like.
The pyrrolopyridinium derivatives of the present invention prepared as described above may be purified, for example, by conventional means such as distillation, chromatography, recrystallization, etc. The pyrrolopyridinium derivatives of the present invention may be readily identified by conventional means such as NMR, mass analysis, fluorescence spectroscopy, etc.
In manufacturing an antibody of the pyrrolopyridinium derivatives of the present invention, a derivative of the present invention is polymerized with a carrier, for example, bovine serum albumin (BSA) or limpet blood dye protein (KLH), using a conventional method to make a hapten, for example a method such as that used to make an antibody to pentosidine as described in the article (Clin. Chem., 40/9, 1766-1773 (1994)). The product hapten is then used in a conventional immunization method, such as immunizing a mouse, rabbit, or the like. Following the immunizing step, a polyclonal antibody or a monoclonal antibody of the pyrrolopyridinium derivative of the present invention can be prepared by conventional methods.
The pyrrolopyridinium derivatives of the present invention can be measured via a conventional method such as ELISA or immunological staining using an antibody prepared from a derivative. The pyrrolopyridinium derivatives of the present invention can be also measured directly by chromatography, such as HPLC or the like, or by spectroscopy, for example, utilizing their fluorescent wave length.
The amount of the pyrrolopyridinium derivatives of the present invention and antibodies prepared therefrom can be measured and used as an index for the degree of accumulation of Advanced Glycation Endproducts in vivo, whereby the progress of diabetes mellitus, diabetic complications, renal failure, dialysis-related complications, amyloidosis, aging or diseases accompanied by aging can be evaluated. Also, the produced amount and the accumulated amount of the pyrrolopyridinium derivatives of the present invention and antibodies prepared therefrom can be measured in the presence and absence of a test drug to evaluate the pharmaceutical effectiveness of a test drug used to treat diabetes mellitus, diabetic complications, dialysis-related complications, amyloidosis, aging or diseases accompanied by aging.
Substrates to be measured for the pyrrolopyridinium derivatives of the present invention and antibodies prepared therefrom include, for example, living tissues, living tissue extracts, body fluid and/or urine; preferable examples of substrates may include human skin tissues, human skin tissue extracts, human lens extracts, human arteriosclerotic tissues, renal tissues of patients suffering from diabetic nephropathy, tissues of aged people, cerebral tissues of patients suffering from Alzheimer disease, human blood, human plasma, human serum and human urine; and, particularly preferable examples of such substrates may include, human skin tissues, human skin tissue extracts, human plasma, human serum and human urine.
Preferred embodiments of the present invention are as follows.
(1) Pyrrolopyridinium derivatives represented by the above-mentioned formulae (I), (II) or (III) and pharmaceutically acceptable salts thereof.
(2) An antibody prepared from the pyrrolopyridinium derivatives according to subparagraph (1) useful as an antibody against Advanced Glycation Endproducts and against the pyrrolopyridinium derivatives of the present invention which are produced in the body.
(3) A method for the diagnosis of diabetes, diabetic complications, dialysis-related complications, amyloidosis, aging and diseases accompanied by aging comprising using the pyrrolopyridinium derivatives according to subparagraph (1) as an indicator.
(4) A method for the diagnosis of diabetes, diabetic complications, dialysis-related complications, amyloidosis, aging and diseases accompanied by aging comprising using the antibody according to subparagraph (2) in an assay as an antibody against Advanced Glycation Endproducts and against the pyrrolopyridinium derivatives of the present invention which are produced in the body.
(5) A method for diagnosis according to subparagraphs (3) or (4) comprising measuring the pyrrolopyridinium derivatives of the present invention in a living tissue, an extract from the living tissue, body fluid and/or urine.
(6) A method for diagnosis according to subparagraph (5) comprising measuring the pyrrolopyridinium derivatives of the present invention in body fluid and/or urine.
(7) A method for diagnosis according to subparagraph (6) wherein an extract from body fluid and/or urine is human blood, human plasma, human serum or human urine.
(8) A method for diagnosis according to subparagraph (5) comprising measuring the pyrrolopyridinium derivatives of the present invention in a living tissue and/or an extract from the living tissue.
(9) A method for diagnosis according to subparagraph (8) wherein an extract from a living tissue and/or an extract from the living tissue is human skin tissues or human skin tissue extracts.
(10) A method for diagnosis comprising measuring the pyrrolopyridinium derivatives of the present invention to evaluate the production and/or the accumulation of Advanced Glycation Endproduct(s) in vivo according to any of subparagraphs (3) to (9).
(11) A method for evaluating of the effectiveness of pharmaceuticals such as therapeutic agents for diabetes, those for diabetic complications, those for dialysis-related complications, those for amyloidosis, preventive agents for aging and therapeutic agents for diseases accompanied by aging comprising using the pyrrolopyridinium derivatives according to subparagraph (1) as an indicator.
(12) A method for evaluating of the effectiveness of pharmaceuticals such as therapeutic agents for diabetes, those for diabetic complications, those for dialysis-related complications, those for amyloidosis, preventive agents for aging and therapeutic agents for diseases accompanied by aging comprising using the antibody according to subparagraph (2) to evaluate the production and/or the accumulation of Advanced Glycation Endproduct(s) in vivo.
The present invention is further illustrated by the following non-limiting examples wherein all parts, percentages and ratios are by weight, all temperatures are in xc2x0 C., and all reactions are conducted at about atmospheric pressure and room temperature unless indicated to the contrary: